Role of Δ133p53 in Tumor Necrosis Factor-induced survival of p53 functions in MKN45 gastric cancer cell line.

OBJECTIVE: To explore the role of Δ133p53 in the effect of recombinant mutant human Tumor Necrosis Factor (rmhTNF) on two gastric cancer cell lines. MATERIALS AND METHODS: MKN45 (with Δ133p53 expression) or SGC7901 (without Δ133p53 expression) cells were treated with rmhTNF of different concentrations only or combined with fluorouracil (5-FU), and the growth inhibition rate was detected by a cell counting kit, and apoptosis by flow cytometry. The mRNA of Δ133p53, p53, Gadd45α, MDM2, PTEN and Bax was measured by reverse transcription PCR (RT-PCR) or Nested PCR (nPCR). RESULTS: On Δ133p53-positive MKN-45 cells, the effect of rmhTNF was significant in growth inhibition test (t = -9.558, p < 0.01); also, the effect of 5-FU was improved by rmhTNF with remarkable time- and dose-effect (F = 82.742, p < 0.01; F = 128.583, p < 0.01). However, on Δ133p53-negative SGC-7901 cells, no growth inhibition was showed by rmhTNF only (t = -0.121, p > 0.05). In apoptosis test, the effect of rmhTNF was significant on MKN45 cells, and the effect of 5-FU was improved significantly by rmhTNF (F = 123.931, p < 0.05). In mRNA measurement, rmhTNF-induced up-regulation of p53 accompanied with down-regulation of Δ133p53, which correlated significantly to the change of p53 downstream molecules, including MDM2, PTEN, Gadd45α, and Bax. CONCLUSIONS: The results in these experiments suggested that Δ133p53 play a pivotal role in rmhTNF-induced survival of p53 functions in Δ133p53-positive MKN-45 cells.

Polymorphism of flagellin A gene in Helicobacter pylori.

AIM: To study the polymorphism of flagellin A genotype and its significance in Helicobacter pylori (H. pylori). METHODS: As the template, genome DNA was purified from six clinical isolates of H. pylori from outpatients, and the corresponding flagellin A fragments were amplified by polymerase chain reaction. All these products were sequenced. These sequences were compared with each other, and analyzed by software of FASTA program. RESULTS: Specific PCR products were amplified from all of these H. pylori isolates and no length divergence was found among them. Compared with each other, the highest ungapped identity is 99.10%, while the lowest is 94.65%. Using FASTA program, the alignments between query and library sequences derived from different H. pylori strains were higher than 90%. CONCLUSION: The nucleotide sequence of flagellin A in H. pylori is highly conservative with incident divergence. This information may be useful for gene diagnosis and further study on flagellar antigen phenotype.

Relationship between genotype and phenotype of flagellin C in Salmonella.

AIM: To discover the relationship between the genotype and antigen serotype of flagellin C among Salmonella strains. METHODS: Fragment of Salmonella flagellin C in plasmid pLS408 was cloned, sequenced and compared with the corresponding sequence in other strains. Salmonella strains including two typhi strains, one paratyphoid strain, one enteritidis and one typhimurium strain were isolated from outpatients. Genome DNA was purified respectively from these clinical isolates, then the corresponding flagellin C fragment was amplified by polymerase chain reaction,and the amplification products were analyzed by agarose gel electrophoresis. RESULTS: The cloned fragment includes 582 nucleotides encoding the variable region and partial conservative region of Salmonella flagellin C in plasmid pLS408. With comparison to the corresponding sequences reported previously, there is only a little difference from other strains with the same flagellar serotype in both nucleotide and amino acid level. Specific PCR products were amplified in Salmonella strains with flagellar serotype H-1-d including S. muenchen, typhi and typhimurium, but not in S. paratyphoid C or S. enteritidis strains. CONCLUSION: In this experiment, the specificity of nucleotide sequence could be found in flagellin C central variable regions as it exists in flagellar serotypes in Salmonella. It may be helpful to developing a rapid, sensitive, accurate and PCR-based method to detect Salmonella strains with serotype H-1-d.